Ahrenhotz et al., xe2x80x9cA conditional suicide system in Escherichia coli based on intracellular degradation of DNAxe2x80x9d Appl. Environ. Microbiol. 60,3746-3751(1994).
Altenbuchner et al., xe2x80x9cPositive selection vectors based on palindromic DNA sequencesxe2x80x9d Methods Enzymol. 216, 457-466 (1992).
Balbas et al., xe2x80x9cPlasmid vector pBR322 and its special-purpose derivativesxe2x80x94a reviewxe2x80x9d Gene 50, 3-40 (1986).
Bernard et al., xe2x80x9cNew ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markersxe2x80x9d Gene 148, 71-74 (1994).
Bolivar et al., xe2x80x9cConstruction and characterization of new cloning vehicles, II. A multipurpose cloning systemxe2x80x9d Gene 2, 95-113 (1977).
Burns D. M. and Beacham, I. R., xe2x80x9cPositive selection vectors: a small plasmid-vector useful for the direct selection of Sau3A-generated overlapping DNA fragmentsxe2x80x9d Gene 27, 323-325 (1984).
Clark, J. M., xe2x80x9cNovel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerasesxe2x80x9d Nucl. Acids Res. 16, 9677-9686 (1988).
Debarbouille, M. and Raibaud, O, xe2x80x9cExpression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoterxe2x80x9d J. Bacteriol. 153, 1221-1227 (1983).
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The present invention relates to positive selection vectors for direct cloning of PCR-amplified nucleic acids. The invention also deals with modulation of regulatory elements for developing such vectors. The invention greatly reduces, if not eliminates, exonuclease-induced false positive clones in a DNA cloning experiment.
Recent advances in the field of molecular biology and genetic engineering include polymerase chain reaction or PCR as described in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188. To amplify or reproduce copies of a targeted nucleic acid, PCR uses a polymerase, targeted sequence-specific forward and reverse primers, deoxynucleotides and a minute amount of target nucleic acid as the template. Exponential amplification of the targeted DNA sequence is achieved by repeated cycles of denaturation of double-stranded DNA followed by primer annealing and primer extension.
PCR-amplified DNA itself has been used for diagnosis, quantitation of the template DNA, direct sequencing and several other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as detection of polymorphism, mutations, sequencing, expression of genes, synthesis of RNA probes etc., it is often necessary to obtain a large quantity of DNA. This necessitates isolation of a bacterial clone carrying the PCR-generated targeted DNA fragment in a vector. Various strategies have been described for cloning PCR-generated DNA fragments into appropriate vectors. One such method involves incorporation of restriction endonuclease cleavage sites near the 5xe2x80x2 end of the PCR primers. The PCR product thus obtained is subjected to purification, restriction digestion with the respective endonuclease followed by ligation into a compatible vector, transformation and identification of the bacterial clone carrying the PCR fragment (Kaufmann and Evans, 1990, BioTechniques 9, 304-306).
The most commonly used strategy involves the nontemplate-dependent terminal transferase or extendase activity of Taq DNA polymerase, which usually produces a dAMP (deoxyadenosine monophosphate) overhang at the 3xe2x80x2 end of the PCR-amplified DNA fragment (Clark, 1988, Nucl. Acid Res. 16, 9677-9686; Hu, 1993, DNA Cell Biol. 12, 763-770). The PCR product thus obtained is ligated into a linearized vector carrying a dTMP (deoxythymidine monophosphate) overhang at the 3xe2x80x2 end (U.S. Pat. No. 5,487,993; Mead et al., 1991, BioTechniques 9, 657-663; Holton and Graham, 1991, Nucl. Acids Res. 19, 1156). In a similar strategy, Taq DNA polymerase generated PCR fragments carrying dAMP overhang at the 3xe2x80x2 end are ligated into a linearized vector carrying an inosine or uracil overhang at the 3xe2x80x2 end (U.S. Pat. No. 5,856,144).
Since the above-mentioned vectors lack the positive selection capability, upon transformation, all host cells carrying either the recombinant vector (containing an insert) or the non-recombinant vector (containing no insert) grow in the desired medium at an equal growth rate. To differentiate between a host cell carrying the non-recombinant religated vector from the host cell carrying the recombinant vector, the DNA fragment to be cloned is usually inserted into a chromogenic gene, the product of which is thus inactivated rendering the recombinant colony white in a chromogenic medium. When the chromogenic gene is lacZ, the transformant carrying the non-recombinant vector turns blue in the presence of X-gal, the substrate for the lacZ gene product xcex2-galactosidase (Messing et al., 1977, Proc. Natl. Acad. Sci. 79, 3642-3646; Norrander et al., 1983, Gene 26, 101-106; Yanisch-Perron et al., 1985, Gene 33, 103-119). When the number of recombinant colonies are low and non-recombinant colonies are high in a plate, then it becomes very difficult to differentiate the recombinant colonies from the non-recombinant colonies. High number of colonies on a plate also lead to contamination between the recombinant and non-recombinant colonies.
To ameliorate the problems associated with the chromogenic selection of recombinant clones many vectors have been developed with positive selection capability allowing only the recombinant clone to grow in a selection medium. Most of these positive selection vectors have been developed based on insertional inactivation of lethal genes (Bums and Beacham, 1984, Gene 27, 323-325; Balbas et al., 1986, Gene 50, 3-40; Pierce et al., 1992, Proc. Natl. Acad. Sci. 89, 2056-2060; Henrich and Plapp, 1986, Gene 42, 345-349; Henrich and Schmidtberger, 1995, Gene 154, 51-54; Bernard et al., 1994, Gene 148, 71-74; Kuhn et al., 1986, Gene 42, 253-263; U.S. Pat. Nos. 5,910,438; 5,891,687). A vector system based on abolition of sensitivity towards metabolite has also been described (Kast, 1994, Gene 138,109-114). Vectors have also been constructed based on selection by means of DNA-degrading or RNA-degrading enzymes (Yaznin et al., 1996, Gene 169, 131-132; Ahrenhotz et al., 1994, Appl. Environ. Microbiol. 60, 3746-3751) as well as based on selection by destruction of long palindromic DNA sequences (Altenbuchner et al., 1992, Methods Enzymol. 216, 457-466).
The presently available positive selection vectors as well as other cloning vectors have several disadvantages. An inherent problem of a vector with a lethal or a chromogenic gene is a high number of false positive clones, i.e., clones without any insert. The false positive clones could arise as revertants following mutations in the lethal or chromogenic gene rendering it inactive. False positive clones may also arise from transformation of linearized vectors, which may get deleted and subsequently recircularized inside the host cell thus inactivating the lacZ or the lethal gene. However, the biggest disadvantage of every cloning system available today is the generation of exonuclease-induced false positive clones. The reagents used in restriction digestion, PCR and ligation, particularly restriction enzymes, polymerases and ligases, are usually contaminated with exonucleases, which may not be completely removed from larger lots of commercial preparations. Exonuclease digestion deletes some nucleotide bases from the cloning site in the chromogenic or lethal gene in a linearized vector DNA. Thus recircularization of such vectors result in inactivation of the chromogenic or lethal genes, and upon transformation these vectors give false positive transformant clones. Similarly, a palindromic sequence could also be destroyed by exonuclease digestion, thus giving false positive clones.
When a small DNA fragment is inserted in frame with the nucleotide sequence of the lethal gene or the chromogenic gene, then the function of the lethal or chromogenic gene may not be altered, thus making it impossible to clone such small DNA fragments. Furthermore, when cloning of a small DNA fragment results in only diminished function of the lethal gene, then clones grow at a reduced rate in case of positive selection vectors. These clones could be confused with the non-recombinant clones growing because of diminished selection pressure due to, for example, long period of incubation.
A further disadvantage of the vectors based on lethal genes is that sometimes a complex medium is required to activate the selection mechanism (Kast, 1994, Gene 138, 109-114). Also, the positive selection vectors carrying lethal or chromogenic genes require special host cells for transformation, e.g., CcdB based vectors require Fxe2x88x92 host cells (U.S. Pat. No. 5,910,438), CAP based vectors require adenyl-cyclase positive host cells (U.S. Pat. No. 5,891,687) and lacZ based vectors require lacxe2x88x92 host cells (Messing et al., 1977, Proc. Natl. Acad. Sci. 79, 3642-3646; Norrander et al., 1983, Gene 26, 101-106; Yanisch-Perron et al., 1985, Gene 33, 103-119). A special regulatory system, usually lacI or CI repressor system (U.S. Pat. No. 5,910,438; Pierce et al., 1992, Proc. Natl. Acad. Sci. 89, 2056-2060), has also to be in place preventing the expression of the lethal gene in the host cell used for large scale preparation of the vector DNA.
The object of the present invention is to develop a simple cloning and/or a sequencing vector which should have the capability of positive selection allowing only the recombinant clones (carrying an insert DNA) to grow in a selection medium, whereas, the non-recombinant clones (carrying no insert) will not grow. A major object of the present invention is to eliminate or greatly reduce the false positive clones associated with all the presently available cloning systems. Especial emphasis is placed on the elimination of exonuclease-induced false positive clones. Thus it is aimed to apply the principle of modulation of a regulatory element, which involves insertional reconstruction of a regulatory element controlling transcription, translation, DNA replication and termination. It was decided to develop a positive selection vector based on insertional reconstruction of a regulatory element of an antibiotic resistance reporter gene lacking the said regulatory element. When the reporter gene is an antibiotic resistance gene, after reconstruction of its regulatory element, upon transformation of a host cell the antibiotic resistance reporter gene is expressed thus allowing only the clones making the antibiotic resistance reporter protein to grow in a specific selection medium containing the respective antibiotic.
Use of the principle of reconstruction of a regulatory element should also greatly reduce, if not eliminate, revertants because firstly, probability of spontaneous mutational creation of a regulatory element is minimal, and secondly, any spontaneous mutation in the coding sequence of an antibiotic resistance reporter gene would most probably destroy the function of the reporter gene protein resulting in inhibition of the growth of the host cell in the selection medium containing the respective antibiotic.
A vector system based on antibiotic resistance gene as the reporter gene should also eliminate the need for any special type of host cells.
Elimination of the disadvantages associated with presently available vectors is a desirable objective, and hence the present invention will be a substantial technological achievement.
The present invention describes a strategy for developing positive selection vectors based on regulatory element modulation, wherein such modulation is achieved via reconstruction or destruction of a regulatory element controlling transcription, translation, DNA replication and termination. The invention also describes the use of such vectors for direct cloning of PCR products. As an example of application of the strategy a positive selection vector pREM5Tc has been developed based on reconstruction of a regulatory element of a reporter gene. The vector pREM5Tc carries a functionally inactive mutant xe2x88x9235 region of the E. coli promoter, a transcriptional regulatory element. The xe2x88x9235 region of the tetracycline resistance gene promoter has been changed to 5xe2x80x2-AAACCC-3xe2x80x2, whereas, the consensus xe2x88x9235 region of an E. coli promoter is 5xe2x80x2-TTGACA-3xe2x80x2, and is situated 17+/xe2x88x921 basepairs (bp) upstream of the xe2x88x9210 region of a promoter (Harrley and Renolds, 1987, Nucl. Acid Res. 15, 2343-2361). Even though the xe2x88x9210 region of the tetracycline resistance gene promoter in pREM5Tc has been converted into a consensus one of 5xe2x80x2-TATAAT-3xe2x80x2 (Harrley and Renolds, 1987, Nucl. Acid Res. 15, 2343-2361) the above change in the xe2x88x9235 region of the promoter stops transcription from this promoter, and as a result the vector upon transformation of a host cell is unable to produce tetracycline resistance gene protein, and hence cannot confer resistance to tetracycline and does not grow in a medium containing tetracycline. A unique cloning site Sma I (5xe2x80x2-CCCGGG-3xe2x80x2) has been created, and the axis of symmetry of this palindrome (5xe2x80x2-CCCGGG-3xe2x80x2) is located just 17 bp upstream of the xe2x88x9210 region (5xe2x88x92-TATAAT-3xe2x80x2) of the tetracycline resistance gene promoter. Upon Sma I restriction cleavage blunt-ended linearized vector is generated, and hence if any DNA fragment carrying 5xe2x80x2-TTGACA-3xe2x80x2 at its 3xe2x80x2 end is inserted in this position, the xe2x88x9235 region of the tetracycline resistance gene promoter is reconstructed resulting in a recombinant plasmid which upon transformation should confer resistance to tetracycline.
A PCR primer carrying the nucleotides 5xe2x80x2-TGTCAA-3xe2x80x2 at its 5xe2x80x2 end is used in PCR. The resulting blunt-ended PCR products thus obtained would carry 5xe2x80x2-TTGACA-3xe2x80x2 at the 3xe2x80x2 end of the strand complementary to the primer. Ligation of this PCR product into the above-mentioned Sma I digested vector reconstructs the xe2x88x9235 region of 5xe2x80x2-TTGACA-3xe2x80x2, which works as a functional xe2x88x9235 region when the recombinant vector transforms a host cell thus expressing the tetracycline resistance gene and conferring resistance to tetracycline. This also ensures unidirectional cloning of the insert.
The upstream region of the xe2x88x9235 region of tetracycline resistance gene in this vector has been changed so that it should not reconstruct a functional xe2x88x9235 region even upon exonuclease digestion without destroying the regulatory elements of the selectable marker ampicillin resistance gene. Thus this cloning system greatly reduces false positive clones induced by exonuclease digestion. The region between the start codons of ampicillin and tetracycline resistance genes has also been modified such that it does not carry any other consensus xe2x88x9210 region for the tetracycline resistance gene, except the recognized consensus xe2x88x9210 region situated 14 bp downstream of the Sma I cloning site.
In between the start codons for the tetracycline and ampicillin resistance genes some restriction sites, e.g., Sac I, Sac II, Not I and Sfi I sites, have been introduced for an easy extraction of the insert DNA fragment.